羊乳和牛乳理化特性比较及其掺假检测研究
发布时间：2019-07-10 09:44
【摘要】：乳制品的质量与安全问题的核心就是控制好乳源。本课题对羊乳和牛乳的蛋白质含量及组分、酒精稳定性和凝乳特性做了比较研究，并且以牛α-乳白蛋白和α-酪蛋白为目标蛋白，建立了可以快速、灵敏、准确地检测羊乳中掺入牛乳含量的直接竞争ELISA法。 羊乳和牛乳理化特性比较研究。以脱脂的羊乳和牛乳为样品，通过凯氏定氮法和SDS-聚丙烯酰胺凝胶电泳测定羊乳和牛乳的蛋白质含量及其蛋白质组分。测得，羊乳的蛋白质含量高于牛乳的，分别为（3.43±0.06）g/100mL和（3.19±0.07）g/100mL（置信度95%）；两者的蛋白组分也存在一定的差异，通过图谱可以看出，牛乳的酪蛋白中以α-酪蛋白含量最多，而羊乳中以β-酪蛋白含量最多。准备原料乳、巴杀乳和冷冻复融乳乳样，测得羊乳乳样的酒精稳定性值依次为46±1、42±2、40±1（%,V/V），凝乳时间依次为297±2、310±2、266±2（S）；牛乳乳样的酒精稳定性值依次为78±1、78±1、78±1（%,V/V）；凝乳时间依次为571±3、590±1、485±1（S）。实验证明，羊乳的胶体稳定性比牛乳的差。 兔抗牛α-乳白蛋白和α-酪蛋白抗体的制备与特性研究。以牛α-乳白蛋白和α-酪蛋白为抗原，免疫新西兰大白兔，采集高效价血清。依次采用饱和硫酸铵分级沉淀法和蛋白A亲和色谱法纯化血清抗体，得到纯化兔抗牛α-LA-IgG和α-CN-IgG样品。效价均可达到1:16000，蛋白质含量分别为0.35mg/mL，0.43mg/mL，纯度分别为88.31%、89.23%。硫氰酸铵洗脱法测得α-LA-IgG和α-CN-IgG对α-LA的亲和力指数分别为2.6mol/L、1.1mol/L，对α-CN的亲和力指数分别为1.6mol/L、2.5mol/L。anti-α-LA-IgG特异性很好，anti-α-CN-IgG与κ-CN和β-CN存在一定交叉反应，两种抗体均与羊乳存在一定的交叉反应。经抗体修饰技术处理后，这些交叉反应均明显降低。 牛乳α-乳白蛋白和α-酪蛋白的酶标记。选用辣根过氧化物酶，通过过碘酸钠氧化法分别标记α-乳白蛋白和α-酪蛋白。结果显示，当HRP与α-LA的分子个数比为1.5:1时，标记效果比较好（结合率25.4%，，标记率85.7%）；α-CN的分子个数比也为1.5:1时，标记效果比较好（结合率43.8%，标记率84.49%）。经ELISA检测，酶标抗原的效价分别可以达到1:320和1:640。 直接竞争ELISA方法的建立。棋盘滴定法确定抗体最佳包被浓度（anti-α-LA-IgG和anti-α-CN-IgG分别为1:160,1:320）和酶标抗原最佳稀释浓度（都为1:20）。确定封闭液1%G的体积为200μL，封闭时间为1h。脱脂乳的最佳稀释度为1:80。用anti-α-LA-IgG和anti-α-CN-IgG分别建立直接竞争ELISA法检测羊乳中掺入的牛乳含量。经评价指标分析，该方法的检测线性范围都为0%~50%（V/V），最低检测限分别为2.5%和4%，变异系数小于5%。 本研究通过羊乳和牛乳的理化特性及其胶体稳定性的比较，为科学合理地利用羊乳，生产具有独特功能的羊乳制品提供了理论依据；建立的基于牛乳α-LA和α-CN的直接竞争ELISA检测法，特异性、重复性、敏感性都比较高，可以作为检测羊乳及其制品中掺入牛乳检测的技术手段。
文内图片：
图片说明：亚胶束模型中酪蛋白胶束的结构
[Abstract]:The core of the quality and safety of dairy products is to control the milk source. The protein content and composition, alcohol stability and curd property of goat milk and milk were compared and studied. And the direct competition ELISA method for accurately detecting the milk content of the goat milk. Comparative Study on the Physical and Chemical Properties of Goat Milk and Milk The protein content of goat milk and milk and its protein group were determined by the method of Kjeldahl and SDS-polyacrylamide gel electrophoresis. The protein content of goat milk was higher than that of cow's milk (3.43-0.06) g/100 mL and (3.19-0.07) g/100 mL (95% confidence). in addition, in that goat milk, the content of the HCO3-casein is the most The alcohol stability of milk-like milk samples was 46,1,42,2,40,1 (%, V/ V), and the time of curd was 297, 2,310, 2,266 and 2 (S). The alcohol stability of milk-like milk was 78%,78%,78%,1 (%, V/ V). ); the time of the curd is 571-3,590-1,485-1 (S The experimental results show that the colloidal stability of the goat milk is higher than that of the milk. The preparation of rabbit anti-bovine serum-milk albumin and the anti-bovine-casein antibody and the preparation of the same Study on the sex of New Zealand white rabbits with bovine serum-milk albumin and calcium-casein as antigen, and high efficiency the serum antibody is purified by adopting a saturated sulfuric acid precipitation method and a protein A affinity chromatography in sequence, and the purified rabbit anti-bovine serum-LA-IgG and the antigen-CN-Ig are obtained The titer of G samples can reach 1:16000, the protein content is 0.35 mg/ mL, 0.43 mg/ mL, and the purity is 88.31%,89. The affinity index of L-LA-IgG and HCO3-CN-IgG to the antigen-LA was 2.6 mol/ L, 1.1 mol/ L, 1.6 mol/ L and 2.5 mol/ L respectively. The reaction of the two kinds of antibodies has a certain relation with the goat milk. The cross reaction is clear after treatment with the antibody modification technology. significantly reduced milk-milk-milk albumin and egg-casein White enzyme marker. Horseradish Peroxidase is used, and the sodium peroxide-milk albumin is respectively marked by the sodium periodate oxidation method. The results showed that when the molecular weight ratio of HRP to LA-LA was 1.5:1, the labeling effect was good (the binding rate was 25.4%, the labeling rate was 85.7%), and the number of the molecular number of the E-CN was also 1.5:1, the labeling effect was good (the binding rate was 43.8%, the labeling rate was 84). .49%). The titer of the enzyme-labeled antigen can be 1:320 and 1:320, respectively. 1:640. Direct competition ELI The best coating concentration of the antibody was determined by the checkerboard titration (1:160,1:320) and the optimal concentration of the enzyme-labeled antigen (1:160,1:320) and the best dilution of the enzyme-labeled antigen (1:160,1:320, respectively). the volume of the blocking solution 1% g was determined to be 200. m u.l, The closed time is 1 h. The best dilution of skim milk The release was 1:80. The direct competitive ELISA was used to detect the goat milk with anti-antigen-LA-IgG and anti-antigen-CN-IgG, respectively. The detection linear range of the method is 0% ~ 50% (V/ V) and the minimum detection limit is 2.5% and 4%, respectively. Through the comparison of the physical and chemical properties and the colloidal stability of the goat milk and the milk, this study provides a theoretical basis for the scientific and reasonable utilization of the goat milk and the production of the sheep milk products with unique functions; and the establishment of the direct competition ELI based on the bovine milk-LA and the L-CN The SA detection method is high in specificity, repeatability and sensitivity, and can be used as a detection goat milk and a product thereof.
【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2512521
文内图片：
图片说明：亚胶束模型中酪蛋白胶束的结构
[Abstract]:The core of the quality and safety of dairy products is to control the milk source. The protein content and composition, alcohol stability and curd property of goat milk and milk were compared and studied. And the direct competition ELISA method for accurately detecting the milk content of the goat milk. Comparative Study on the Physical and Chemical Properties of Goat Milk and Milk The protein content of goat milk and milk and its protein group were determined by the method of Kjeldahl and SDS-polyacrylamide gel electrophoresis. The protein content of goat milk was higher than that of cow's milk (3.43-0.06) g/100 mL and (3.19-0.07) g/100 mL (95% confidence). in addition, in that goat milk, the content of the HCO3-casein is the most The alcohol stability of milk-like milk samples was 46,1,42,2,40,1 (%, V/ V), and the time of curd was 297, 2,310, 2,266 and 2 (S). The alcohol stability of milk-like milk was 78%,78%,78%,1 (%, V/ V). ); the time of the curd is 571-3,590-1,485-1 (S The experimental results show that the colloidal stability of the goat milk is higher than that of the milk. The preparation of rabbit anti-bovine serum-milk albumin and the anti-bovine-casein antibody and the preparation of the same Study on the sex of New Zealand white rabbits with bovine serum-milk albumin and calcium-casein as antigen, and high efficiency the serum antibody is purified by adopting a saturated sulfuric acid precipitation method and a protein A affinity chromatography in sequence, and the purified rabbit anti-bovine serum-LA-IgG and the antigen-CN-Ig are obtained The titer of G samples can reach 1:16000, the protein content is 0.35 mg/ mL, 0.43 mg/ mL, and the purity is 88.31%,89. The affinity index of L-LA-IgG and HCO3-CN-IgG to the antigen-LA was 2.6 mol/ L, 1.1 mol/ L, 1.6 mol/ L and 2.5 mol/ L respectively. The reaction of the two kinds of antibodies has a certain relation with the goat milk. The cross reaction is clear after treatment with the antibody modification technology. significantly reduced milk-milk-milk albumin and egg-casein White enzyme marker. Horseradish Peroxidase is used, and the sodium peroxide-milk albumin is respectively marked by the sodium periodate oxidation method. The results showed that when the molecular weight ratio of HRP to LA-LA was 1.5:1, the labeling effect was good (the binding rate was 25.4%, the labeling rate was 85.7%), and the number of the molecular number of the E-CN was also 1.5:1, the labeling effect was good (the binding rate was 43.8%, the labeling rate was 84). .49%). The titer of the enzyme-labeled antigen can be 1:320 and 1:320, respectively. 1:640. Direct competition ELI The best coating concentration of the antibody was determined by the checkerboard titration (1:160,1:320) and the optimal concentration of the enzyme-labeled antigen (1:160,1:320) and the best dilution of the enzyme-labeled antigen (1:160,1:320, respectively). the volume of the blocking solution 1% g was determined to be 200. m u.l, The closed time is 1 h. The best dilution of skim milk The release was 1:80. The direct competitive ELISA was used to detect the goat milk with anti-antigen-LA-IgG and anti-antigen-CN-IgG, respectively. The detection linear range of the method is 0% ~ 50% (V/ V) and the minimum detection limit is 2.5% and 4%, respectively. Through the comparison of the physical and chemical properties and the colloidal stability of the goat milk and the milk, this study provides a theoretical basis for the scientific and reasonable utilization of the goat milk and the production of the sheep milk products with unique functions; and the establishment of the direct competition ELI based on the bovine milk-LA and the L-CN The SA detection method is high in specificity, repeatability and sensitivity, and can be used as a detection goat milk and a product thereof.
【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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